The Caliciviridae is a family of positive-strand RNA viruses and now consists of 11 genera: Norovirus, Sapovirus, Bavovirus, Lagovirus, Minovirus, Nacovirus, Nebovirus Recovirus, Salovirus, Valovirus, and Vesivirus. The diseases caused by caliciviruses vary, according to the virus and its host species. Members of the Caliciviridae causing diarrheal disease in humans belong to the genera Norovirus and Sapovirus. The Caliciviruses Section (CS) in LID conducts research related to caliciviruses, with an emphasis on the noroviruses because of their predominant role in human diarrheal disease. Noroviruses are genetically-diverse and most human norovirus pathogens belong to either Genogroup I (GI) or Genogroup II (GII), with GII.4 as the predominant genotype. Our laboratory has a long-term interest in tracking the epidemiology of these viruses, beginning with the discovery of Norwalk virus, the prototype strain, by Dr. Al Kapikian in 1972. We have an ongoing epidemiological investigation of noroviruses in immunocompromised patients enrolled in research protocols at the NIH Clinical Research Center. Our goal is to understand the genetic diversity of the viruses associated with chronic norovirus infection, and how they adapt to become chronic. We developed virus-matched probes for the evaluation of norovirus cell tropism in patient endoscopic biopsies, and showed that epithelial cells (and not immune cells) in the small intestine are the primary target cells for human norovirus replication. In tandem, we expanded our work in the human enteroid 3-dimensional cell culture systems and successfully applied single cell RNA sequencing to better understand the replication of human norovirus in permissive epithelial target cells. Our work continued on approaches to develop immunotherapy. We continued mapping B cell epitopes in the norovirus capsid and optimizing methods to perform high-throughput screening for antibody specificity profiling. In collaboration with investigators at Children's National Medical Center in Washington, D.C., we began investigation of potential T cell epitopes conserved among norovirus strains. This work overlaps with our epidemiological surveillance studies in immunocompromised patients, and we are defining the genetic stability over time of B and T cell epitopes that have potential for broad functional activity. We completed siRNA functional screens with NCATs using the highly permissive murine norovirus model system. We developed a number of CRISPR/cas9-modified cell lines to validate promising hits from the screen for therapeutic investigations, and the mechanistic analyses are underway.